Holliday junctions (HJs) are four-way DNA structures that occur in DNA repair by homologous recombination. Specialized nucleases, termed resolvases, remove (i.e., resolve) HJs. The bacterial protein RuvC is a canonical resolvase that introduces two symmetric cuts into the HJ. For complete resolution of the HJ, the two cuts need to be tightly coordinated. They are also specific for cognate DNA sequences. Using a combination of structural biology, biochemistry, and a computational approach, here we show that correct positioning of the substrate for cleavage requires conformational changes within the bound DNA. These changes involve rare high-energy states with protein-assisted base flipping that are readily accessible for the cognate DNA sequence but not for non-cognate sequences. These conformational changes and the relief of protein-induced structural tension of the DNA facilitate coordination between the two cuts. The unique DNA cleavage mechanism of RuvC demonstrates the importance of high-energy conformational states in nucleic acid readouts.
- MeSH
- arginin chemie MeSH
- bakteriální proteiny chemie MeSH
- biokatalýza MeSH
- DNA bakterií chemie metabolismus MeSH
- křížová struktura DNA chemie MeSH
- párování bází MeSH
- sekvence nukleotidů MeSH
- simulace molekulární dynamiky MeSH
- Thermus thermophilus metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus-Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- DNA vazebné proteiny chemie genetika metabolismus MeSH
- konzervovaná sekvence MeSH
- molekulární evoluce * MeSH
- stabilita proteinů MeSH
- Thermus thermophilus genetika metabolismus MeSH
- vazba proteinů MeSH
- vysoká teplota * MeSH
- Publikační typ
- časopisecké články MeSH
